An essential step in metastatic dissemination of tumor cells involves intravasation where the carcinoma cell must cross the basement membrane of the duct/acinus and then the basement membrane of the endothelium of blood vessels. The ability to degrade and penetrate the dense extracellular matrix of a basement membrane is important for transmigration in both cases. Degradation and penetration of basement membranes is generally believed to require protrusive activity of tumor cells, in particular, the formation of invadopodia and lamallipodia, which are regulated by EGF receptor and tyrosine kinase signaling. Invadopodia are protrusions of the tumor cell membrane with associated protease activity. The ability to make invadopodia is correlated with metastatic potential in tumor cells. To identify the mechanisms regulating the steps to invadopodium maturation, and thereby understand how invadopodia are involved in both chemotaxis and invasion of tumor cells, during the previous funding period we studied the initiation and maturation of invadopodia in detail. During these studies we identified 4 stages of formation of an invadopodium: precursor formation; activation of actin polymerization; stabilization and ECM degradation. During progression through these stages, key regulatory events were identified including: 1. N-WASp activation dependent recruitment of cortactin; 2. the regulation of cofilin activity by binding of cofilin to cortactin; 3. Cofilin and N-WASp dependent actin polymerization; 4. Arg-dependent cortactin phosphorylation which regulates #s 1-3; 5. Rho-family G-protein dependent regulation of N-WASp, cofilin and degradation of ECM. In this application we propose to study each of these regulatory events, and the mechanisms by which they are choreographed, during invadopodium formation and cancer cell migration. PUBLIC HEALTH RELEVANCE: Metastasis is the primary cause of death for cancer patients. Escape from the primary tumor and invasion into a new tissue depends on the ability of cancer cells to migrate. The initial step in breast carcinoma cell invasion involves tumor cells crossing the basement membrane of the mammary duct/acinus. An essential step in metastatic dissemination of tumor cells involves intravasation where the carcinoma cell must again cross the basement membrane of the endothelium of blood vessels. Degradation and penetration of basement membranes is generally believed to require protrusive activity of tumor cells, in particular the formation of invadopodia and lamallipodia. In this application we propose to study the regulation and function of invadopodia in breast tumor cells.